Development of nanocellulose scaffolds with tunable structures to support 3D cell culture

Carbohydrate Polymers 148 (2016) 259–271

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Development of nanocellulose scaffolds with tunable structures to support 3D cell culture Jun Liu a,∗ , Fang Cheng b,c , Henrik Grénman d , Steven Spoljaric e , Jukka Seppälä e , John E. Eriksson b,c , Stefan Willför a , Chunlin Xu a,∗ a

Johan Gadolin Process Chemistry Centre, c/o Laboratory Wood and Paper Chemistry, Åbo Akademi University, Porthansgatan 3, Åbo/Turku, 20500, Finland Department of Biosciences, Åbo Akademi University, Turku, 20520, Finland c Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, 20521, Finland d Johan Gadolin Process Chemistry Centre, Laboratory of Industrial Chemistry and Reaction Engineering, Åbo Akademi University, Biskopsgatan 8, Åbo/Turku, 20500, Finland e Polymer Technology, Department of Biotechnology and Chemical Technology, Aalto University School of Chemical Technology, P.O. Box 16100, Aalto, 00076, Finland b

a r t i c l e

i n f o

Article history: Received 3 December 2015 Received in revised form 2 April 2016 Accepted 14 April 2016 Keywords: Hydrogel Aerogel Cell culture Nanocellulose Scaffold Tissue engineering

a b s t r a c t Swollen three-dimensional nanocellulose films and their resultant aerogels were prepared as scaffolds towards tissue engineering application. The nanocellulose hydrogels with various swelling degree (up to 500 times) and the resultant aerogels with desired porosity (porosity up to 99.7% and specific surface area up to 308 m2 /g) were prepared by tuning the nanocellulose charge density, the swelling media conditions, and the material processing approach. Representative cell-based assays were applied to assess the material biocompatibility and efficacy of the human extracellular matrix (ECM)-mimicking nanocellulose scaffolds. The effects of charge density and porosity of the scaffolds on the biological tests were investigated for the first time. The results reveal that the nanocellulose scaffolds could promote the survival and proliferation of tumor cells, and enhance the transfection of exogenous DNA into the cells. These results suggest the usefulness of the nanocellulose-based matrices in supporting crucial cellular processes during cell growth and proliferation. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Traditional tissue engineering techniques which rely on harvesting autologous, allogenic or xenogenic tissues or organs encountered various serious problems, such as donor shortage, undesirable immune responses, and the risk of pathogen transmission (Burg, Porter, & Kellam, 2000; Ko, Sfeir, & Kumta, 2010). The current trend in tissue engineering for replacing or restoring damaged or diseased tissue is the use of naturally derived three-dimensional (3D) scaffolds or matrices which support crucial cellular activities, such as cell attachment, proliferation, and subsequent tissue formation (Czaja, Young, Kawecki, & Brown, 2007; Joshi et al., 2016; Moroni, de Wijn, & van Blitterswijk, 2006). The challenge has been in finding optimal matrices that can act as cell carriers as well as signal providers for tissue growth and regeneration and that are able to generate tissues and organs possessing biological structures and functions (Balakrishnan & Banerjee, 2011). Application of 3D natural polymer-based scaffolds,

∗ Corresponding authors. E-mail addresses: jun.liu@abo.fi (J. Liu), cxu@abo.fi (C. Xu). http://dx.doi.org/10.1016/j.carbpol.2016.04.064 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

such as cellulose-based hydrogel and aerogel, in tissue engineering as a template to support crucial cellular activities has been increasingly developed (Atila, Keskin, & Tezcaner, 2015; Joshi et al., 2016; Pei et al., 2015). A 3D cellular model based on cellulose can mimic various cellular features and functions in the extracellular matrices (ECM) in vivo, such as cell–cell communication, gene expression, and biological responsiveness (Neves et al., 2015). To accurately reproduce the characteristics of human cancer tissues for investigating cancer migration, tumor cell cycle progression and intercellular communication, human cancer cells such as the epithelial-derived Hela cells and hematopoietic-derived Jurkat cells have been applied in the in vitro tests of 3D tumor models (Ko et al., 2010; Metaxa, Efthimiadou, & Kordas, 2014; Zhao et al., 2014). Cellulose-based materials, have shown a potential in the abovementioned application owing to their intrinsic characteristics, such as biocompatibility, non-cytotoxicity, tunable 3D architecture and porous microstructures, and desired mechanical properties (Shelke, James, Laurencin, & Kumbar, 2014). For example, 3D cellulose scaffolds and its hybrids with chitosan, alginate, and agarose have been fabricated for various applications in tissue engineering (Ko et al., 2010).

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Lately, nanocellulose materials, which mainly include cellulose nanocrystal (CNC), nanofibrillated cellulose (NFC), and bacterial cellulose (BC), are increasingly applied in biomedical or pharmaceutical areas, thanks to their non-toxic, biocompatible, and cost effective properties (Moon, Martini, Nairn, Simonsen, & Youngblood, 2011). The NFC hydrogel and its resultant aerogel have been developed for biomedical applications, such as 3D stem cell cultures (Bhattacharya et al., 2012; Lou et al., 2014), drug delivery (Valo et al., 2011, 2013), and implantable scaffolds (e.g. prosthetic heart valves, vascular grafts (Cherian et al., 2011), and nucleus pulposus in intervertebral disks (Eyholzer et al., 2011)). So far, most of these researches were based on the NFC suspensions that were produced via a pure mechanical process (e.g. homogenization), and such NFC fibrils bearing fewer negative charge groups on the surface compared with those prepared from the 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) oxidation process. The NFC prepared from the TEMPO/NaClO/NaBr oxidization approach is known for its uniform size distribution (5–10 nm in width) and its negative charge properties. The negative charge on the surface of NFC contributes to the stable dispersion of its suspension in comparison to the CNC suspension which is commonly forming aggregates or precipitates. Furthermore, the NFC suspensions tend to form a hydrogel when the concentration and charge density reach a certain level (e.g. 0.8 mmol/g, 0.5% (Liu, Korpinen, Mikkonen, Willför, & Xu, 2014; Syverud, Pettersen, Draget, & Chinga-Carrasco, 2014). The development of NFC-based cell-compatible materials to mimic the roles of ECM in body tissue enables their biomedical applications such as cell therapy, tissue engineering, wound healing, in vitro diagnostics, and 3D cell cultures (Park, Lee, & Hyun, 2015; Seliktar, 2012). In the tissue engineering application, the in situ formed hydrogels with desired swelling capacity allow them to maintain a moist environment that resembles the hydrated state of the natural tissue in vivo (Hunt, Chen, van Veen, & Bryan, 2014; Rimmer, 2011). Appropriate porous microstructure and interconnected network in the hydrogels allow cells to penetrate through, and is of importance for nutrients and metabolic waste transportation in 3D cell culture (Aegerter, Leventis, & Koebel, 2011; Stella, D’Amore, Wagner, & Sacks, 2010). Thus, tuning the porous microstructure of the NFC-based hydrogel and aerogel was subjected to study in the current work. Moreover, biological responses of cells to foreign materials need to be investigated prior to the potential biomedical applications of NFC-based materials (Aegerter et al., 2011). The cytotoxicity test has proved the non-toxicity of the neat NFC (Alexandrescu, Syverud, Gatti, & Chinga-Carrasco, 2013). However, to the best of our knowledge, no research has been reported to investigate the effect of the chemical properties of NFC-based materials, e.g. surface charge density, on crucial cellular processes in 3D cell culture of cancer cells. Therefore, the present study proposes a novel approach to fine tune the properties of NFC hydrogels and also aerogels, i.e. porosity and surface charge density, to investigate the interrelationship between the materials processing and their structure, properties, and performance. Cell-based assays relevant for 3D tumor model study were performed to investigate the biocompatibility and efficiency of the 3D NFC-scaffolds with epithelial-derived Hela cells and hematopoietic-derived Jurkat cells.

ized water before the reaction. The TEMPO (0.1 mmol/g fiber) and sodium bromide (1.0 mmol/g fiber) were dissolved in 50 mL deionized water and mixed with the fiber suspension, followed by adjusting the pH of the slurry to 10.0 by the addition of 0.5 M NaOH. The oxidation was started by adding the NaClO (10 mmol/g fiber) solution (10% wt. active chlorine) dropwise to the slurry. The total volume of NaClO was added within one-third of the total reaction time to prepare NFC with a different charge density. The pH of the reaction system was maintained at 10.5 by the addition of 0.5 M NaOH during the reaction. TEMPO-oxidized cellulose fibers were precipitated in ethanol with a ratio of 1:3 (v/v) followed by centrifugation. The collected fiber was thoroughly washed with deionized water and separated by centrifugation for three times. The oxidized cellulose at a consistency of ca. 0.5% was fibrillated by a domestic blender (OBH Nordica 6658, Denmark) for 5 min to yield the NFC suspensions. The charge density (carboxylate content) of the NFC fibers was determined in suspension by conductometry titration (Liu et al., 2014). In this study, NFC with charge densities of 1.56 ± 0.11 mmol/g and 2.04 ± 0.03 mmol/g were used and coded as LC (low charge) NFC and HC (high charge) NFC, respectively. The NFC suspensions were stored at 4.0 ◦ C before further analysis or processing. The NFC free-standing films were prepared via filtration of the NFC dispersion (0.1%, wt.) on a nylon membrane filter (0.2 ␮m pores, Ø = 90 mm) with a funnel (Sterlitech, USA) under vacuum. Wet pressing (WP) of the filtrate cake was carried out after filtration by transferring the filtrate cake between the filter papers and pressing under a pressure of 88 mbar to blot water. Hot press (HP) treatment of the NFC free-standing films was carried out following the WP preparation but at 80 ◦ C and at a pressure of 2.3 bar (Scan CM 64:00) for 20 min. The thickness of the films was measured using a micrometer (L&W, 975465, Stockholm, Sweden). 2.2. Swelling degree of the NFC free-standing films and the preparation of NFC hydrogels Water uptake of the NFC free-standing films in swelling media to form hydrogels was monitored by measuring the water uptake with a gravimetric method and the size increment (thickness) of the films with a caliper. The NFC films were immersed into the swelling media and soaked at a predetermined temperature (25 ◦ C, 37 ◦ C, 50 ◦ C) and pH (3.0, 7.0, 10.0). At a given time of swelling, the swollen film (hydrogel) was weighed after taking out from the swelling media and removing the excess water from the surface with blotters. The thickness of the hydrogels was measured with a caliper at the end of the swelling experiment. The swelling degree was calculated according to Eq. (1): Swelling degree X = (Xwet − Xdry )/Xdry ,

(1)

Where, X denotes the weight or thickness of the films in wet or dry condition, respectively. The samples were named sequentially according to the charge density of the NFC (low charge and high charge, LC and HC); the NFC film press processing approaches (wet press and hot press, WP and HP); the temperature (25, 37, 50 ◦ C) and pH value (3.0, 7.0, 10.0) of the swelling media. 2.3. Preparation of NFC aerogels

2. Experimental methods 2.1. Preparation of NFC suspension and free-standing films The NFC suspension was prepared from bleached birch Kraft pulp according to a previously reported method (Liu et al., 2014). In brief, one gram of the fiber was disintegrated in 50 mL deion-

The NFC hydrogels were obtained by swelling the NFC films in the swelling media at the predetermined temperature and pH for 24 h. Solvent exchange of the NFC hydrogels was carried out step-wise with ethanol (25%, 50%, 75%, 95%, and 99.5%) for 24 h in each step, followed by solvent exchange from ethanol to tertbutanol for 24 h in three steps. The samples were frozen using liquid

J. Liu et al. / Carbohydrate Polymers 148 (2016) 259–271

nitrogen for 30 min followed by freeze-drying for 72 h at a temperature of −45 ◦ C and under a pressure of 0.1 mbar. The effects of the NFC charge density, hot and wet press, temperature and pH of the swelling media on the swelling properties and porosity of the aerogels were studied by preparing aerogels under each condition. 2.4. Specific surface area, apparent density, and pore size distribution of NFC aerogels

digital caliper (Fowler Pro-Max, USA). At least five parallel hydrogel and aerogel specimens were measured for each sample, and the results were calculated and reported as mean ± S.D. The elastic modulus of the hydrogel and aerogel was obtained by extrapolating and linear fitting of the elastic region of the stress-strain curves (Ohya, Nakayama, & Matsuda, 2001). 2.9. Cell culture

The specific surface area (SSA) of the NFC aerogels was determined by N2 adsorption using a Sorptomatic 1900 (Carlo erba instruments, UK), and calculated by the Brunauer-Emmett-Teller method (Brunauer, Emmett, & Teller, 1938). The mesopore specific volume was calculated by the Barrett-Joyner-Halenda method (Barrett, Joyner, & Halenda, 1951). The aerogel samples were degassed and dried at 120 ◦ C for 3.0 h prior to measurement. The apparent density (␳* ) of the aerogels was determined by measuring the mass (±0.01 mg) and the geometric dimensions of the samples, the porosity of the aerogels was calculated according to Eq. (2). The skeletal density of cellulose (␳c = 1.50 g/cm3 ) used in the nitrogen adsorption measurements was verified employing Helium pycnometry. Porosity = (1 − ␳∗ /␳c ) × 100%

261

(2)

2.5. Scanning electron microscope imaging The NFC aerogels were mounted on a sample stage with conductive double-side sticking tape and coated with carbon by an ion sputter coater. Images of the NFC aerogels were captured using a scanning electron microscope (LEO Gemini 1530, Germany) equipped with an UltraDry Silicon Drift Detector (Thermo Scientific, USA) at an accelerating voltage of 5 kV. 2.6. Transmission electron microscopy (TEM) imaging TEM images of NFC were obtained using a JEM-1400 Plus TEM instrument (JEOL, Japan) as previously reported (Bober et al., 2014). The size dimension of the NFC was analyzed using the iTEM program (Olympus Soft Imaging Solutions, Germany) from the NFC’s TEM images.

Cell culture on the standard platform (24-well plate) and NFC matrices (film and aerogel) was carried out using epithelialderived Hela cells and hematopoietic-derived Jurkat cells for in vitro assessment of the common cellular functions (e.g. cell attachment, viability, and proliferation on scaffolds) (Mitra, Sailakshmi, Gnanamani, & Mandal, 2013). Sterilization of all the NFC films and aerogels was carried out by exposing to ultraviolet radiation for 30 min. Hela cells and Jurkat cells were seeded onto the NFC matrices by dropping the cell suspension on the film or aerogel surface at a seed density of 5 × 105 cells per piece film (5 × 5 mm × 45 ␮m, length × width × thickness) or aerogel in 24-well plate. The cell suspension was slowly absorbed into the matrix’s interior due to the inherent hydrophilicity of the NFC, meanwhile the matrix started to swell to form hydrogels. Hela cells and Jurkat cells were seeded on a Mock platform and used as control. Hela cells were maintained with DMEM (4.5 mM glucose) supplemented with 2 mM l-glutamine, 100 IU/mL penicillin and streptomycin, and 10% heat-inactivated FBS (Invitrogen). Jurkat cells were cultured in RPMI medium with the same supplements as for the Hela cells. All cells were incubated with 5% CO2 at 37 ◦ C and 95% humidity. The medium was renewed daily for three days. 2.10. Cells viability assay and fluorescence-activated cell sorting (FACS) analysis Cells that were incubated with NFC matrix or the control Mock platform were collected after 72 h incubation and then analyzed for cell viability. For the detection of permeabilized (dead) cells, the cells were trypsinized and resuspended in 50 ␮g/mL Propidium Iodide (PI) (Sigma-Aldrich) in PBS for 10 min at room temperature. The samples were then analyzed by FACSCalibur flow cytometer (FSC, BD Pharmingen). Trypan Blue analysis was used to prove the FACS results.

2.7. Fourier transform infrared spectroscopy (FTIR) analysis 2.11. Cell proliferation assay Before the measurement, the NFC suspension was treated with 0.1 M HCl for 3.0 h to acidify the carboxylates to their protonated form, and freeze-drying was carried out (Fujisawa, Okita, Fukuzumi, Saito, & Isogai, 2011). The FTIR spectra of the NFC samples were recorded using an FTIR spectrometer (a Bruker ALPHA series) using the ALPHA platinum in ATR module in the range of 400–4000 cm−1 with a 4 cm−1 resolution, and with an accumulation of 64 scans. 2.8. Mechanical properties of NFC hydrogels and aerogels The mechanical properties of the NFC hydrogels and their resultant aerogels were characterized using a TA Instrument Dynamic Mechanical Analyzer Q800 (New Castle, USA) operated in compression mode. The cylindrical hydrogel (ø = 12.85, mm) or the cubic aerogel (3 × 3 × thickness, mm) were tested at a compression rate of 1.0 N/min by using load cells of 5.0/10 N. A preload of 0.05 N was applied before compression testing. The compression tests were terminated when the normal force reached the maximum loading capacity, or when the slide reached the limit distance. The initial dimensions of the hydrogels and aerogels were measured using a

For in vitro proliferation assays, cells were seeded on the NFC matrices in 96-well cell plates and placed in the integrated incubator on Cell IQ live cell imaging and automated analysis system instruments (CM Technologies, Tampere, Finland). The samples were imaged automatically at regular 6 h intervals with kinetic proliferation data for 72 h. Quantification of the number of live, dead, and dividing cells was extracted from the phase contrast image series and proliferation videos were made based on the corresponding data extraction. 2.12. Confocal imaging of cells on the matrices Cells retrieved from the NFC matrices were fixed with 3.7% paraformaldehyde for 30 min. After antigen retrieval with 0.1 M citrate buffer (pH 6.0), sections were blocked with 5% normal goat serum, stained with primary antibodies for overnight at 4 ◦ C, secondary antibodies for 3 h at room temperature, and counterstained with DAPI for 10 min. Confocal images were acquired at room temperature using Zeiss Zen software on a Zeiss LSM780 confocal laser scanning microscope (Carl Zeiss, Inc.) with Plan-Apochromat 10×

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3. Results and discussion

Fig. 1. Swelling kinetics (weight increment, wt.) of NFC films as a function of time. Charge density of HC and LC NFCs were 2.04 ± 0.03 mmol/g and 1.56 ± 0.11 mmol/g, respectively.

(NA 0.45, air). The following fluorochromes were used: Alexa Fluor 488, Alexa Fluor 546, and DAPI (405).

Since the use of native ECM in tissue engineering applications has been restricted by the potential pathogen transmission and the host immunogenic response (Stella et al., 2010), the development of ECM-like scaffolds for tissue engineering applications is of importance. To mimic the cell attachment and proliferation in the 3D ECM scaffold in vivo for potential 3D cell culture application and tumor study, this work proposed a novel approach to encapsulate and distribute the cells in the formed NFC matrices, and the encapsulated cells are supposed to grow and proliferate along the NFC matrices’ 3D network as in vivo. Since, the cellular functions are affected by the physical, chemical and structural properties of the matrix, chemical, physical, and mechanical approaches were carried out to tailor the matrix with desired properties for potential 3D cellular model study and 3D scaffold construction in tissue engineering. As shown in the scheme in Fig. S1, the NFC films were prepared by filtration and different pressing methods. The corresponding aerogels were prepared using the solvent exchange and freeze-drying approach from the NFC hydrogels which were formed after swelling of the NFC films in swelling media. A series of material process methods and parameters were applied to tune the structure, mechanical strength, and thus the potential biocompatibility of the NFC-based matrices. 3.1. Swelling behavior of the NFC films

2.13. Reverse transfection efficiency and expression of fluorescent proteins The red fluorescent proteins (RFP) were used to investigate the Hela cells reverse transfection efficiency in the 3D NFC matrices. For one well, 5 × 105 cells per piece film or aerogel were mixed with Lipofectamine LTX reagent (Invitrogen) containing 1 ␮g RFP plasmid in 24-well plate. The RFP protein expression was monitored under a fluorescent microscope. The experiments were performed a few days post transfection. 2.14. Statistical analysis The cell culture results are expressed as the mean ± S.E. Comparisons between 2 groups were analyzed by 2-tailed t-tests. Comparisons between multiple groups were analyzed by 1-way ANOVA. P < 0.05 was considered significant. Statistical differences were calculated with the two-tailed unpaired t-test and differences were considered significant at p ≤ 0.05. All other results were reported as mean ± S.D.

Water retention is a key parameter for the ECM to maintain extracellular homeostasis (Frantz, Stewart, & Weaver, 2010). The swelling kinetics of the NFC films in water at room temperature were monitored during swelling by a gravimetric test and depicted in Fig. 1. The films from both the LC and HC NFCs swelled rapidly in 10.0 h, and the swelling reached equilibrium after 24.0 h. However, the films from HC NFC had swollen much faster than that of the films from LC NFC as indicated by the higher slope of the swelling curve of HC NFC before 10.0 h. The maximum swelling degree of the films from LC and HC NFCs was 122 and 447, respectively, indicating that the thin films of LC and HC NFCs can absorb and retain 122 and 447 times of water of their own weight, respectively. The equilibrium swelling capacity of a polyelectrolyte network hydrogel, like the NFC in this case, depends on both the intrinsic parameters of the material and the environmental conditions (e.g. pH, ionic strength, and solvent composition) (Rimmer, 2011). During the swelling process, the ionic charge on the NFC backbone induces a Gibbs-Donnan effect and the subsequent osmotic effect, which allows more water to penetrate into the hydrogel to dilute the con-

Fig. 2. The effect of the swelling media temperature (A) and pH (B) on the swelling degree (weight increment, wt.) of the NFC films. The samples were named sequentially according to the charge density of the NFC (low charge and high charge, LC and HC) and the NFC film press processing approaches (wet press and hot press, WP and HP).

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Fig. 3. The effect of the swelling media temperature and pH on the density (A, C) and porosity (B, D) of the NFC aerogels.

Table 1 Specific surface area (SSA), pore specific volume (PSV), density, and porosity of the NFC aerogels.

b

HC-WP−25 HC-WP-50 HC-WP−25 (pH3)c HC-HP-25 LC-WP-25 LC-WP-50 LC-WP−25 (pH3)c LC-HP-25

SSA (m2 /g)a

PSV (cm3 /g)

Density (kg/m3 )

Porosity (%)

308.0 297.6 279.4 267.4 242.1 203.6 157.5 175.9

0.73 0.83 0.96 0.86 0.42 0.46 0.49 0.70

6.4 ± 0.5 4.2 ± 0.2 95.0 ± 2.9 8.8 ± 0.3 17.8 ± 3.7 16.7 ± 3.8 179.6 ± 21.4 18.5 ± 1.3

99.6 ± 0.03 99.7 ± 0.01 93.5 ± 0.19 99.4 ± 0.02 98.8 ± 0.24 98.9 ± 0.25 88.0 ± 1.43 98.8 ± 0.09

Values were calculated using the skeletal density ␳c = 1.50 which was measured by Helium pycnometry. The samples were named sequentially according to the charge density of the NFC (low charge and high charge, LC and HC); the NFC film press processing approaches (wet press and hot press, WP and HP); the temperature (25, 37, 50 ◦ C) and pH value (3.0, 7.0, 10.0) of the swelling media. c Samples were swollen in a media with a pH of 3.0. If not specified, other samples were swollen in a neutral environment. a

b

centration of the charges inside the network and thus promote the network swelling. The electrostatic repulsion between the charges promotes the hydrogel to swell by expanding the macromolecular network. The interconnected channels in the hydrogels network may promote the further swelling of the hydrogels by capillary forces and convection of water into the porous hydrogel. The significant difference in the swelling degree of the LC and HC NFC films (Fig. 1) suggests the key role of the charge density of the NFC in contribution to the swelling degree of the films. Therefore, the selection of the charge density of the NFC is critical to control the swelling behavior and the subsequent volume change of the hydrogels, and to tune the porous structure of the resultant aerogels. The films prepared from the HC NFC showed much higher swelling degree in both weight (Fig. 2A) and volume (thickness increment, Fig. S4A) than that of the LC NFC at all the temperatures investigated. The swelling degree of the films, especially the

HC films, increased with the raise of temperature. Hot press treatment slightly decreased the swelling degree of the LC NFC films (up to 26%, LC-HP-37 vs LC-WP-37, Fig. 2A), but sharply decreased that of the HC NFC films (up to 50% HC-HP-25 vs HC-WP-25, Fig. 2A). However, the elevated temperature from 25 ◦ C to 37 ◦ C, and from 37 ◦ C to 50 ◦ C can help to recover part of the lost swelling degree (ca. 10% in each stage) of the high-charged NFC films caused by the hot press treatment. Both the films from LC and HC NFCs barely swelled in the swelling media at a pH value of 3.0 (Figs. 2 B and S4B), while the swelling degree of all the NFC films slightly decreased in the media at a pH value of 10.0 compared to the neutral swelling media. As discussed above, the swelling behavior of the NFC hydrogels is determined by the environmental conditions and the intrinsic parameters of the NFC. The NFC possesses weak carboxylic acid groups (see the FTIR spectra at 1728 cm−1 in Fig. S3, (Fujisawa

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Fig. 4. SEM images of the NFC aerogels processed at different conditions. The samples were named sequentially according to the charge density of the NFC (low charge and high charge, LC and HC); the NFC film press processing approaches (wet press and hot press, WP and HP); the temperature (25, 37, 50 ◦ C) and pH value (3.0, 7.0, 10.0) of the swelling media. If not specified, samples were swollen in a neutral environment (pH 7.0). NOTE, the scale bars of images for LC and HC aerogels are different. The two images at the bottom are the higher magnification view of the samples above.

et al., 2011) which has a pKa of 3.0–5.0, and it is known to be fully dissociated at around pH 8.5 (Junka, Filpponen, Lindström, & Laine, 2013; Spaic, Small, Cook, & Wan, 2014). The electrostatic repulsion between the ionic charges (−COOH) on the nanofibrils changed at different pH, which caused the dissociation or association of the carboxylic acid groups. Consequently, the changed repulsion resulted in the difference in the swelling behavior of the films in media with different pH values (Fig. 2B). The decreased swelling degree at pH 10.0 (Fig. 2B) might be due to the increased ionic strength at corresponding pH value (more counterions of

Na+ ), which reduced the electrostatic repulsion by neutralizing part of the fixed charges on the nanofibrils and also diminished the Donnan effect by decreasing the ion species concentration difference between the gel inside and the external solution. Such pH-responsive swelling behavior of the materials shows promising approach to tune the 3D microstructure of the materials for desired biomedical application, e.g. for the controlled release of bioactive agents and for monitoring of relevant cellular processes in tissue engineering (Chinga-Carrasco & Syverud, 2014).

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Table 2 Mechanical properties of the NFC hydrogels and the resultant aerogels. Sample

Compressive Stress (kPa)

Strain (%)

Modulus (kPa)

Hydrogel

HC-WP−25a HC-WP-50 HC-WP−25 (pH3)b HC-HP-25 LC-WP-25 LC-WP-50 LC-WP−25 (pH3)b LC-HP-25

4.1 ± 0.5 1.1 ± 0.2 11.1 ± 3.8 4.4 ± 0.2 7.5 ± 0.4 5.2 ± 0.6 16.0 ± 0.9 10.4 ± 1.0

71.4 ± 8.0 54.5 ± 7.7 71.6 ± 5.8 73.6 ± 3.2 63.0 ± 3.6 57.8 ± 4.3 86.4 ± 9.5 76.0 ± 6.8

9.6 ± 1.3 3.0 ± 0.4 35.5 ± 6.7 10.8 ± 0.8 27.6 ± 4.1 23.1 ± 2.10 33.3 ± 6.7 30.6 ± 5.9

Aerogel

HC-WP-25 HC-WP-50 HC-WP−25 (pH3)b HC-HP-25 LC-WP−25c LC-WP−50c LC-WP−25 (pH3)b , c LC-HP−25c

28.0 ± 6.6 34.2 ± 3.1 35.7 ± 5.4 31.8 ± 4.9 60.3 ± 5.9 94.4 ± 14.9 104.41 ± 2.76 62.80 ± 5.10

195.8 ± 10.5 251.8 ± 21.1 198.1 ± 13.7 239.5 ± 5.8 91.8 ± 7.8 186.7 ± 11.0 179.9 ± 7.6 156.4 ± 3.3

24.7 ± 0.7 28.5 ± 3.4 47.2 ± 3.6 26.5 ± 1.5 65.5 ± 3.5 100.0 ± 6.2 88.1 ± 5.5 66.9 ± 4.0

a The samples were named sequentially according to the charge density of the NFC (low charge and high charge, LC and HC); the NFC film press processing approaches (wet press and hot press, WP and HP); the temperature (25, 37, 50 ◦ C) and pH value (3.0, 7.0, 10.0) of the swelling media. b Samples swelled in media with a pH of 3.0. If not specified, other samples swelled in a neutral environment. c Values were obtained from the measurement of two overlapped specimens to reach the thickness limit (ca. >1.0 mm) of the DMA measurement. If not specified, other samples were determined individually.

Besides the key intrinsic chemical properties of the materials, wet press and hot press were also applied to tune the structure properties of the NFC materials. Hot press treatment was earlier developed as a rapid method to prepare NFC films with excellent mechanical strength (Osterberg et al., 2013). In the present work, as shown in Figs. 2 and S4, hot press treatment can be used to lower the swelling degree of the NFC films by forming the irreversible hydrogen bonds between cellulose fibrils or causing the so-called hornification, especially for the highly charged NFC films. The increase of swelling temperature can accelerate the movement of the water molecules, consequently leads to a higher swelling degree (Fig. 2A) of the NFC films. However, not all the hydrogen bonds between the nanofibrils can be recovered by hydrogen bonds between the water molecules during swelling even at an elevated temperature. The tunable swelling capacity of NFC matrix by judicious controlling of the material intrinsic properties and processing approaches can potentially be used to load them with pharmaceutical compounds or biomolecules like proteins, bioactive polysaccharides, or cells for biomedical and pharmaceutical applications. In this work, the tumor cells (Hela and Jurkat cells) were supposed to be absorbed into the networks of the hydrogels during swelling, meanwhile the cells distribute and grow in the NFC scaffold as in the ECM in vivo. More importantly, the NFC films with desired swelling capacity enable the efficient preservation of a moist environment that resembles the highly hydrated state of the native tissue in vivo (Cushing & Anseth, 2007; Cutting, 2003; Rimmer, 2011; Winter, 1962). 3.2. Pore parameters and pore size distribution of the NFC aerogels ECM-mimicking scaffolds with appropriate pore parameters is critical for cell penetration, cell migration, cellular ingrowth, nutrients transportation, and removal of metabolic waste (Stella et al., 2010). The effects of NFC charge density (LC vs HC), NFC film press treatment (WP vs HP), and the hydrogel swelling environment (temperature and pH) on the density/porosity, and BET specific surface area (SSA) of the aerogels are shown in Fig. 3 and Table 1, respectively. The SEM images show the cross-section (Figs. 4 and S5-3) and surface (Fig. S5-1, S5-2 and S5-3) of the aerogels. As shown in Fig. 3, the density and porosity values of the aerogels from the LC NFC were in the range of 16.8–18.5 kg/m3 (Fig. 3A)

and 98.8–98.9% (Fig. 3B) respectively, suggesting that both the swelling media temperature and the hot press treatment did not significantly affect the LC density and porosity of the NFC aerogels. However, the effect of those treatments was more significant for the HC NFC aerogels. Generally, with the increase of the swelling media temperature, the density (Fig. 3A) of the aerogels decreased, while the porosity (Fig. 3B) increased. The hot press treatment caused higher density and lower porosity of the HC NFC aerogels at all conditions. As shown in Figs. 2 and 3, the aerogel density and porosity highly depend on the swelling degree of the NFC films under different temperature, i.e. the higher the swelling degree of the films is (Fig. 2), the higher the porosity of the aerogel is (Fig. 3). As shown in Fig. 3C, after swelling of the NFC films (LC and HC) in the media with a pH value of 3.0, the resultant aerogels showed 10–15 times higher density compared to that of the aerogels prepared in the media with pH 7.0 and pH 10.0. The hot press treatment resulted in a further increase in the density of the aerogels. Consequently, the porosity of the aerogels decreased by 10–18% (pH 3.0 vs pH 7.0) for the LC aerogels and 5–6% (pH 3.0 vs pH 7.0) for the HC aerogels. Shrinkage of all hydrogels during the solvent exchange and of all aerogels during freeze-drying was observed (Table S1). The shrinkage of the hydrogels and aerogels was mainly affected by the charge density of the NFC, and by the material processing steps (swelling temperature, pH, and hot/wet press). In general, the shrinkage (both after solvent exchange and freeze-drying) values were proportionally related to the swelling degree of the hydrogel. Solvent exchange at low temperature (−20 ◦ C) followed by supercritical CO2 drying method was suggested as a better drying approach to preserve the porous structure and to minimize shrinkage during various aerogels preparations (Aegerter et al., 2011). However, shrinkage of the polysaccharides-based (starch and alginate) aerogels during solvent exchange (23–55%) and supercritical drying (23–36%) still cannot be avoided (Mehling, Smirnova, Guenther, & Neubert, 2009). Therefore, the freeze-drying approach used in the present study might still be acceptable regarding the preservation (final shrinkage 36.1–65.8%, Table S1) of the porous structure of the materials. The SSA values (157–308 m2 /g, Table 1) of the NFC aerogels are in agreement with those reported for NFC aerogels prepared using solvent exchange and freeze-drying (153–284 m2 /g) (Sehaqui, Zhou, & Berglund, 2011). Higher SSA of the NFC aerogels (up to 482 m2 /g) has been reported when the supercritical CO2 drying method was used (Sehaqui, Zhou, Ikkala, & Berglund, 2011). The

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Fig. 5. Prototyping of NFC matrices to support cellular activities in 3D tumor cellular model and tissue engineering. (A) Hela cells and Jurkat cells were incubated with the NFC matrices for 72 h. After incubation, the percentage of dead cells was detected by PI staining and flow cytometry analysis. ± s.e.m.; n = 4. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (B and C) Representative phase contrast images and growth curves of Hela cell lines which were incubated with the NFC matrices for 72 h ± s.e.m.; n = 3. Scale bar, 50 ␮m. Wet pressed high and low charge NFC films (HC-WP and LC-WP), and their resultant aerogels (HC-WP-25 and LC-WP-25) were used for testing.

SSA and pore specific volume (PSV) values of the HC samples were in the range of 267–308 m2 /g and 0.7–1.0 cm3 /g, respectively, while these values of the LC samples were in the range of 157–242 m2 /g and 0.4–0.7 cm3 /g. The apparent density of the LC aerogel samples showed much higher values than that of the HC samples at all conditions, while the porosity values were the opposite. After the hot press treatment, the SSA values of the aerogels of HC and LC NFCs decreased by 13.2% (HC-WP-25 vs HC-HP-25) and 27.3% (LC-WP-25 vs LC-HP-25), respectively. However, the PSV values of both HC and LC aerogels that underwent hot press treatment were slightly increased. The swelling media temperature negatively correlated with the apparent density and SSA values of both the HC and LC aerogels, while the porosity values increased with the increase of the swelling temperature from 25 ◦ C to 50 ◦ C. The decreased swelling pH (pH 3.0) showed a similar effect as the increase of the swelling temperature, however, the increased swelling pH (i.e. 10) did not affect the pore properties of the aerogels significantly (data not shown).

As can be observed in Table 1, the PSV values showed an inverse response compared to SSA values. Similar inverse response has been found between SSA and pore size of the NFC aerogels (Silva, Habibi, Colodette, Elder, & Lucia, 2012). The increased swelling temperature adversely affects the SSA of both the HC and LC NFC aerogels. This might be due to the increased crosslinking between the nanocellulose chains via hydroxyl-carboxyl interaction and hydrogen bonding with the increase of the swelling temperature (Park, Okano, & Ottenbrite, 2010). Consequently, such crosslinking resulted in a dense and rigid network (confirmed by the SEM images in Fig. 4, and compressive stress data in Table 2), which lowered the SSA of the aerogel. With the decrease in pH of the swelling media, the disassociation of the carboxylic acid decreased, consequently, the swelling degree of the films decreased as discussed above. And due to less carboxylic acids that need to be transferred to the non-ionized form compared with the HC NFC, the SSA values of the aerogels decreased signifi-

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Fig. 6. Representative confocal images of the expression of Ki67 (in red) and cytoskeleton marker actin (in green) in Hela cells growing into HC and LC films (A) and aerogels (B) after 72 h incubation. Matrices and nucleus were counterstained with DAPI (blue). Scale bar, 200 ␮m. Wet pressed high and low charge NFC films (HC-WP and LC-WP), and their resultant aerogels (HC-WP-25 and LC-WP-25) were used for testing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

cantly with the decrease in the pH of swelling media (Table 1), and the open-pore structures were diminished (Fig. 4). Although there is no significant difference between the porosity values of the aerogels (<2%, Fig. 3B), except the acidified aerogels (pH 3.0, Fig. 3D), notable volume change was observed during experiments and as verified in the SEM images in Fig. 4. In general, the aerogels from the HC NFC showed more ordered and welldistributed open-pore networks compared to the LC aerogels at the same conditions. The open pore size in both the LC and HC aerogels ranged from 10 to 200 ␮m in diameter. The thick walls (LC-WP-25 and HC-WP-25) of the pores turned into thin layers after the hot press treatment (LC-HP-25 and HC-HP-25), but the openpore structure was less changed. The aerogels of LC-WP-25-pH3 and HC-WP-25-pH3, which were prepared in a media with pH 3.0, lost most of the open pores, especially for the LC aerogels which collapsed into a thinner film. The aerogels of LC-WP-50 and HCWP-50 visually formed dense and thick layers and almost lost the open-pore network, however the higher magnification images (at the bottom of Fig. 4) revealed that the dense walls actually consisted of fine nanofibrillar network structures and fine pores. Most of the SEM images of the aerogel surfaces appeared rough and compact (Fig. S5-1, S5-2), however, the images of the surfaces of HC-WP-50

and LC-WP-50 showed irregular grooves and open-pore fine structures, respectively. A summary of SEM images of the NFC aerogels (medium charge density, wet pressed, swollen in neutral media at r.t.) was presented in Fig. S5-3, which shows both the detailed open-pore networks of the cross-section and the surface, and the fine nanofibrillar networks. As shown in the SEM images in Fig. 4 and Table 1, control of the charge density of the NFC and processing (i.e. wet or hot press treatment, and swelling media conditions) of the materials enables the production of the NFC aerogels possessing a wide array of pore sizes, structures, and morphologies. The tunable open-pore structures or fine fibrous micro- and nano-architectures can completely meet the demand in pore parameters in native ECM (50–500 nm) (Stella et al., 2010). Therefore, the NFC matrix with highly porous morphologies and appropriate pore size distribution may provide a 3D environment to support efficient cell proliferation and growth.

3.3. Mechanical properties of the NFC hydrogels and aerogels The ECM-mimicking scaffolds should provide sufficient mechanical integrity to withstand the dynamic environment upon tissue engineering applications. The mechanical properties of NFC

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Fig. 7. (A) Representative phase contrast images and RFP fluorescent images of Hela cells in different matrices transfected with DNA vectors encoding full length RFP (the total DNA per transfection was equalized with the control vector). Scale bar, 50 ␮m. (B) The quantitation of RFP positive cells per microscopic field. ± s.e.m.; n = 4. ***, p < 0.001. Wet pressed high and low charge NFC films (HC-WP and LC-WP), and their resultant aerogels (HC-WP-25 and LC-WP-25) were used for testing.

hydrogels and the resultant aerogels are summarized in Table 2, and the representative stress-strain curves of the NFC hydrogel and aerogel are presented in Fig. S6. In the compression test, as shown in Fig. S6, the aerogels underwent elastic deformation, plastic deformation (compaction), and densification stages (Aegerter et al., 2011), while the last stage was not observed for the hydrogels due to the collapse of the structures. As shown in Table 2, the overall compressive stress and modulus of the HC hydrogels were lower than those of the LC hydrogels. The charge density of the NFC significantly influences the swelling degree of the hydrogels as depicted in Fig. 1. However, the super high swelling degree (e.g. HC-WP-50 with a swelling degree of 516) resulted in a flexible hydrogel with lower mechanical strength (HC-WP-50 with a compressive stress of 1.12 kPa, Table 2) due to the dramatically plasticizing effect of the hydrogels by water. This intimate relation between mechanical strength and swelling degree of the hydrogels has been summarized elsewhere (Anseth, Bowman, & Brannon-Peppas, 1996). The elevated swelling temperature (from 25 ◦ C to 50 ◦ C) resulted in the decrease in compressive stress and modulus of both HC (HC-WP-25 vs HC-WP-50) and LC (LC-WP-25 vs LC-WP-50) hydrogels. The hot press treatment prior to swelling increased 8.6% of the compressive stress of the HC hydrogel (HC-WP-25 vs HC-HP-25), while the compressive stress of the LC hydrogels enhanced 39.1% after hot press treatment (LCWP-25 vs LC-HP-25). For both the HC and LC samples, the hydrogels that were swollen in the media with a pH of 3.0 (HC-WP-25 (pH3))

and LC-WP-25 (pH3) have much higher mechanical strength than those that were swollen in a neutral environment. Besides the effects of charge density and material processing methods, the physical attribute of the LC and HC NFC fibers (i.e. the aspect ratio difference) might also affect the mechanical strength of the hydrogels and aerogels. As shown in Fig. S2, the LC NFC fibers have higher length and width values than HC NFC ones, indicating that the LC hydrogels and the resultant aerogels might have a more rigid network due to the tangled nanofibrils which contribute to the mechanical strength. The compressive strain of the NFC aerogels in Table 2 indicates the ductile properties of the materials, suggesting that the aerogels can be compressed to a large strain range (91%-251%) depending on the original thickness. It is difficult to make a conclusion of the effect of the NFC charge density on the mechanical properties of the aerogels as shown in Table 2. This is because the mechanical strength of aerogel depends on its structure and density (see Table 1) (Aegerter et al., 2011), and also due to the minor difference of the measurement method for LC (measured for two overlapped specimens) and HC aerogels (measured individually). However, as shown in Table 2, the hot press treatment (HC/LC-WP-25 vs HC/LC-HP-25) and higher swelling temperature (HC/LC-WP-25 vs HC/LC-WP-50) led to a higher compressive stress and modulus for both the HC and LC aerogels. Besides, the NFC aerogels that were prepared at high swelling temperature showed higher mechanical strength (HC-WP-50 vs HC-WP-25 and LC-WP-50 vs LC-WP-25, Table 2). This might be due to the formation of a dense and rigid

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NFC network (SEM images in Fig. 4) by crosslinking between the nanocellulose chains via hydroxyl-carboxyl interaction and hydrogen bonding at a higher swelling temperature (Park et al., 2010). Therefore, all these results suggest that the mechanical strength of NFC hydrogels and aerogels can be tuned using the NFC with appropriate chemical (charge density) and physical (aspect ratio) properties, and by controlling the processing parameters without introducing any toxic chemicals. 3.4. NFC matrices supporting survival, proliferation and exogenous DNA transfer in 3D tumor cellular models and tissue engineering To mimic the 3D ECM scaffolds for 3D cell culture study and for tissue engineering application, the biocompatibility and efficacy of the NFC-based matrices should be assessed (Neves et al., 2015). Therefore, the first goal was to test the functionality of the generated matrices in supporting cell survival. The second goal was to assess the capability of the generated matrices in promoting cell proliferation, which is critical in tissue engineering and tumor study. The epithelial-derived Hela cells and hematopoieticderived Jurkat cells, two cell models which were widely used for tumor studies, were applied to assess the biocompatibility of the NFC-based matrices (films of HC-WP and LC-WP; aerogels of HCWP-25 and LC-WP-25). The cells viability and proliferation in the NFC matrices, and the related conformal images are depicted in Figs. 5 and 6, respectively. Reverse transfection of DNA vectors and fluorescent protein (RFP) expression in the Hela cells in the NFC matrices are shown in Fig. 7. Hela cells and Jurkat cells have been recently used in the study to construct 3D tumor models to accurately reproduce the characteristics of human cancer tissues for investigating cancer migration, tumor cell cycle progression and intercellular communication (Gao, Konno, & Ishihara, 2014; Huang, Qu, Liu, & Chen, 2014; Ikeda et al., 2008; Kurihara & Nagamune, 2005; Nelson-Rees & Flandermeyer, 1976; Zhao et al., 2014). In addition, Hela cells are big adherent cells and Jurkat cells are small suspension cells, which are quite different from each other. It is nice to see both can fit the NFC-based matrices. After 72 h incubation of cells in the existence of NFC matrices, cells were extracted and the percentage of dead cells was determined using Propidium Iodide (PI) staining followed by FACS analysis. We found that both the NFC films and aerogels could support the survival of Hela cells and Jurkat cells, and no clear cell toxicity was observed after 72 h with less than 5% cell death in all settings (Fig. 5A). Interestingly, the NFC films seemed to be more protectant from cell death than the aerogels and the control condition (mock, Fig. 5A). Besides, lower cell death was observed for the LC matrices (both films and aerogels) compare to the HC ones. The non-toxicity of the NFC-based matrix against other cell lines, e.g. the fibroblasts cells (3T3 cells) (Alexandrescu et al., 2013), and the liver progenitor cells (HepaRG cells) (Malinen et al., 2014), has been reported recently. The lower cell death (Fig. 5A) and better cell proliferation results (Fig. 5C) in the NFC film matrices compared to the aerogel matrices might be due to the difference of cell penetration and distribution in the 3D networks of the matrix. For the film matrices, cells were absorbed and penetrated into the interior networks or pores (Fig. 4 and Fig. S5-3) with water due to the Donnan effect and the subsequent osmotic effect during the film swelling (Alberts, 2002; Rimmer, 2011). Therefore, the cells can evenly distribute, grow, and proliferate inside. However, the osmotic effect may be weaker in the network of aerogel, thus the cells may not efficiently attach onto the matrix and evenly distribute inside the network in comparison to the film matrix. This can be confirmed by the confocal imaging analysis of the cell proliferation in the matrices in Fig. 6. The cells are evenly distributed in the 3D networks formed from the films, and the apparent cell density in the film matrices

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is much higher than that in the aerogel matrices. Moderate surface charge of the matrices has been identified to play an important role in the cell biological response and might enhance the cell growth and proliferation (Cartmell, Thurstan, Gittings, Griffiths, Bowen & Turner, 2014; Itoh, Nakamura, Nakamura, Shinomiya, & Yamashita, 2006), which has also been confirmed by the better cell viability and proliferation of the LC film matrices in this work (Fig. 5A and B). However, as shown in Figs. 5A, B, and 6, the higher surface charge of the matrices may hinder the cell biological response and result in slower growth and a higher cell death rate. Representative phase contrast images of NFC matricessupporting cell proliferation are shown in Fig. 5B. Hela cells in both LC and HC NFC films were growing faster than for the control condition (Fig. 5C and supplemental videos), but cells in NFC aerogels were growing slightly slower than for the control (Fig. 5C and supplemental videos) in a 72 h-time-lapse proliferation assay. The confocal imaging analysis (Fig. 6A) of the fixed film matrices after 72 h incubation also suggests that the NFC film matrices displayed a striking induction in proliferating cells into the 3D matrices, as indicated by the intense distribution of cells positive for the proliferation marker, nuclear antigen ki67, as well as cytoskeleton marker actin. The Ki67 and actin positive cell population in aerogels were much smaller (Fig. 6B), supporting the proliferation data in Fig. 5C. This observation further demonstrates that cells growing in NFC film matrices had increased cell proliferation activity. The reverse transfection of DNA vectors and fluorescent protein (RFP) expression is important in the applications of 3D matrix platform for diagnostic and therapeutic purposes. DNA transfer and protein expression of the cells in the NFC film matrices were conducted by using the Red Fluorescent Protein (RFP). As shown in the phase contrast images (Fig. 7A) and the fluorescent imaging analysis (Fig. 7B), the reverse transfection of DNA vectors and the expression of corresponding fluorescent proteins RFP in Hela cells were promoted in the existence of the NFC matrices, especially in the LC matrix as indicated by the intense distribution of RFP proteins. The enhanced cell proliferation rate in the presence of LC matrix can also lead to the reverse transfection promotion (Cartmell et al., 2014). The enhanced DNA transfection and protein expression efficiency in the NFC film matrices, especially the LC film, suggests that the NFC-based matrices can promote not only the normal cell growth and proliferation, but also the transfection of exogenous DNA into the cells. These results suggest that the NFC-based materials are promising candidates as matrices for 3D cell culture study and other tissue engineering applications. Moreover, the charge density of cellulose fibers and the material processing approaches were found to moderately affect the cell proliferation and DNA transfection into the cells.

4. Conclusions NFC films and 3D highly porous aerogels with tunable structures were prepared from wood cellulose towards tissue engineering scaffold and 3D cell culture study by judicious controlling of the NFC intrinsic properties (charge density and aspect ratio) and processing parameters (swelling temperature, pH, and wet and hot press treatments). This work demonstrated a novel approach to encapsulate and distribute the cells in the NFC-based matrices during the swelling process. The NFC 3D matrices, especially the NFC films with low surface charge density, facilitated the encapsulated Hela cells and Jurkat cells for ingrowth, survival, and proliferation. The reverse transfection of exogenous DNA vectors and the expression of fluorescent proteins in Hela cells were promoted in the existence of the NFC matrices. The NFC-based matrices (film, hydrogel, and

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aerogel) with biocompatibility, permeability, reasonable mechanical and chemical stability, and tunable morphology and structure are very promising candidates for 3D cell culture study and the tissue engineering applications. Acknowledgements Jun Liu would like to acknowledge the financial support of the China Scholarship Council and Graduate School of Chemical Engineering. This work is also part of the activities at the Johan Gadolin Process Chemistry Centre, a Centre of Excellence financed by Åbo Akademi University. Dr. Kirsi S. Mikkonen at the University of Helsinki is gratefully acknowledged for valuable discussions. We would like to acknowledge Dr. Markus Peurla from Laboratory of Electron Microscopy, University of Turku, Linus Silvander from Laboratory of Inorganic Chemistry, Åbo Akademi University, and Jyrki Juhanoja from Top Analytica Oy Ab for training of TEM, and SEM imaging. Dr. Holger Wondaczek from Fibre and Cellulose Technology is acknowledged for helping with the FTIR-ART measurement. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2016.04. 064. References Aegerter, M. A., Leventis, N., & Koebel, M. A. (2011). Aerogels handbook. New York: Springer. Alberts, B. (2002). Molecular biology of the cell. New York: Garland Science. Alexandrescu, L., Syverud, K., Gatti, A., & Chinga-Carrasco, G. (2013). Cytotoxicity tests of cellulose nanofibril-based structures. Cellulose, 20(4), 1765–1775. Anseth, K. S., Bowman, C. N., & Brannon-Peppas, L. (1996). Mechanical properties of hydrogels and their experimental determination. Biomaterials, 17(17), 1647–1657. Atila, D., Keskin, D., & Tezcaner, A. (2015). Cellulose acetate based 3-dimensional electrospun scaffolds for skin tissue engineering applications. Carbohydrate Polymer, 133, 251–261. Balakrishnan, B., & Banerjee, R. (2011). Biopolymer-based hydrogels for cartilage tissue engineering. Chemical Reviews, 111(8), 4453–4474. Barrett, E. P., Joyner, L. G., & Halenda, P. P. (1951). The determination of pore volume and area distributions in porous substances. I. Computations from nitrogen isotherms. Journal of the American Chemical Society, 73(1), 373–380. Bhattacharya, M., Malinen, M. M., Lauren, P., Lou, Y. R., Kuisma, S. W., Kanninen, L., et al. (2012). Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture. Journal of Control Release, 164(3), 291–298. Bober, P., Liu, J., Mikkonen, K. S., Ihalainen, P., Pesonen, M., Plumed-Ferrer, C., et al. (2014). Biocomposites of nanofibrillated cellulose, polypyrrole, and silver nanoparticles with electroconductive and antimicrobial properties. Biomacromolecules, 15(10), 3655–3663. Brunauer, S., Emmett, P. H., & Teller, E. (1938). Adsorption of gases in multimolecular layers. Journal of the American Chemical Society, 60(2), 309–319. Burg, K. J. L., Porter, S., & Kellam, J. F. (2000). Biomaterial developments for bone tissue engineering. Biomaterials, 21(23), 2347–2359. Cartmell, S. H., Thurstan, S., Gittings, J. P., Griffiths, S., Bowen, C. R., & Turner, I. G. (2014). Polarization of porous hydroxyapatite scaffolds: influence on osteoblast cell proliferation and extracellular matrix production. Journal of Biomedical Materials Research Part A, 102(4), 1047–1052. Cherian, B. M., Leão, A. L., de Souza, S. F., Costa, L. M. M., de Olyveira, G. M., Kottaisamy, M., et al. (2011). Cellulose nanocomposites with nanofibres isolated from pineapple leaf fibers for medical applications. Carbohydrate Polymer, 86(4), 1790–1798. Chinga-Carrasco, G., & Syverud, K. (2014). Pretreatment-dependent surface chemistry of wood nanocellulose for pH-sensitive hydrogels. Journal of Biomaterials Applications, 29(3), 423–432. Cushing, M. C., & Anseth, K. S. (2007). Materials science. Hydrogel cell cultures. Science, 316(5828), 1133–1134. Cutting, K. F. (2003). Wound exudate: composition and functions. British Journal of Community Nursing, 8(9 Suppl), 4–9. Czaja, W. K., Young, D. J., Kawecki, M., & Brown, R. M., Jr. (2007). The future prospects of microbial cellulose in biomedical applications. Biomacromolecules, 8(1), 1–12. Eyholzer, C., de Couraca, A. B., Duc, F., Bourban, P. E., Tingaut, P., Zimmermann, T., et al. (2011). Biocomposite hydrogels with carboxymethylated, nanofibrillated cellulose powder for replacement of the nucleus pulposus. Biomacromolecules, 12(5), 1419–1427.

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